GZMK Drives Aging-Associated Fibrosis through Complement C3a/C3aR-Dependent Cellular Senescence

Background Aging-related multi-organ fibrosis represents a major clinical challenge, largely due to poorly defined extracellular triggers of cellular senescence and the senescence-associated secretory phenotype (SASP). Granzyme K (GZMK), an immune cell–derived serine protease, is hypothesized to regulate this process through a previously unreported immunoproteolytic cascade. Given the established role of the complement C3a–C3aR axis in tissue aging and fibrosis, we investigated its involvement in GZMK-mediated senescence. Methods We combined transcriptomic analysis of human fibrotic diseases with experimental validation in vitro and in vivo. Primary mouse embryonic fibroblasts (MEFs) were treated with recombinant GZMK to assess senescence and SASP activation. An accelerated aging model was established in C57BL/6 mice using D-galactose, with or without the GZMK inhibitor PPACK. Senescence markers, fibrosis-related proteins, and complement activation were evaluated via Western blot, RT‑qPCR, ELISA, histology, and RNA sequencing. Results GZMK was significantly upregulated in aged and fibrotic human and murine tissues. In MEFs, GZMK triggered cellular senescence, suppressed proliferation, and induced multiple SASP factors. Transcriptomic analysis revealed enrichment in complement pathways, and we demonstrated that GZMK directly cleaves C3 to generate C3a and upregulates C3aR. Both the pro-senescent and pro-SASP effects of GZMK were abolished by C3aR antagonism. In vivo, PPACK treatment alleviated D-gal–induced aging phenotypes, reduced multi-organ collagen deposition, suppressed senescence and local/systemic SASP, and attenuated C3a–C3aR activation. Conclusion Our study identifies a novel GZMK–complement–senescence axis, wherein immune-derived GZMK cleaves C3 to activate C3a–C3aR signaling, thereby driving cellular senescence, SASP, and multi-organ fibrosis. These findings nominate GZMK as a circulating biomarker and a promising therapeutic target for age-related fibrotic diseases.